Salinity tolerance of 47 wild barley genotypes and six barley cultivars was evaluated under control and salinity stress (300 mM NaCl) conditions. Shoot and root dry weight (DW), plant height, membrane stability index (MSI), relative water content, survival rate, leaf malondialdehyde (MDA) and proline contents, root and leaf Na, K, Ca and K/Na ratio, and chlorophyll a fluorescence were measured. Salinity stress caused significant increase in the MDA, proline content, Na and Ca concentrations of the roots and leaves, but resulted in a decrease in the other traits. H. spontaneum genotypes were considerably less affected by the salinity than the genotypes of H. vulgare. Plant survivability was negatively correlated with the Na concentration (r =−.66) but positively correlated with the leaf K/Na ratio (r = .67) and MSI (r = .68).Tolerance mechanisms such as ion exclusion (Na) were likely to be present in the wild barley causing K/Na homeostasis as well as the much lower root and shoot Na, resulting in the higher survival rate. K E Y W O R D S barley breeding, genetic variation, ion content, salt tolerance, wild barley | 305 EBRAHIM Et Al. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Ebrahim F, Arzani A, Rahimmalek M, Sun D, Peng J. Salinity tolerance of wild barley Hordeum vulgare ssp. spontaneum. Plant Breed. 2020;139:304-316.